Fig. 4
DSP107 induces T cell-mediated killing of tumor cells upon cross-linking via CD47 arm of DSP107. A) IL-8 release by HT1080.4-1BB cells triggered by increasing concentrations of DSP107 with CD47-coated plate (dark blue), when plate-bound CD47 is blocked with CD47 mAb (blue), without plate-bound CD47 or with CD47 mAb alone (purple and red), parametric paired t-test was used with respect to control sample (concentration 0) versus treatment with DSP107, B) IL-8 secretion upon treatment of HT1080.4-1BB cells with DSP107 or DSP-MOCK, containing 4-1BBL domain but lacking SIRPα domain, parametric paired t-test was used with respect to control sample treated with DSP107 or DSP-mock or treated sample with plated-bound CD47, C) Binding of DSP107 to CHO-K1 clones expressing low, medium and high levels of CD47 as measured by flow cytometry, parametric paired t-test was used with respect to control sample vs binding of DSP107, D) IL-8 secretion by HT080.4-1BB cells co-cultured with CHO-K1 clones expressing different levels of CD47 upon treatment with DSP107, analyzed as in C, E) Increase of CD25 expression on activated T cells (red diamonds) upon co-stimulation with DSP107 (blue diamonds, left graphs) or CD28 mAb (blue diamonds, right graph), paired t-test was used with respect to control sample (unstimulated) versus treatment with DSP107 or CD28 mAb F) Increase of 4-1BB expression on activated T cells (red diamonds) upon co-stimulation with DSP107 (blue diamonds, left graphs) or CD28 antibody (blue diamonds, right graph), paired t-test was used as in E), G) Representative pictures of co-cultures of SC-1.scFvCD3 with CD3+ (left), CD8+ (middle) and CD4+ (right) T cells, untreated (left picture in each panel) or upon treatment with DSP107 (right picture in each panel), scale bar: 50 μm. H) AnnexinV staining of residual SC-1.scFvCD3 cells in co-cultures with increasing amount of CD3+ (left), CD8+ (middle) or CD4+ (right) without treatment (red) or with 8,4 μg/ml DSP107 (blue), parametric paired t-test was used with respect to control sample (medium control – red) versus treatment with DSP107 (blue) at each E:T ratio. I) T cell-mediated killing of CHOwtscFvCD3 or CHOCD47scFvCD3 with and without DSP107 as measured by flow cytometry, parametric paired t-test was used with respect to control sample vs binding of DSP107. Across the figure, t-test values: n.s. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, all error bars in the figure represent SD